WO2020158183A1 - Bubble discharging method, particle trapping device, and particle analysis device - Google Patents

Abstract

The present invention provides a technique for efficiently discharging bubbles present in a fluid inside a chamber.　Provided is a bubble discharging method comprising: a pressurization step in which positive pressure is applied to a fluid inside a chamber that has at least one well or through hole, contains a microchip, and is partitioned into a first space and a second space; and a valve opening/closing step in which a valve is operated to open and close a first flow channel, which is connected to the first space, and/or a second flow channel, which is connected to the second space.

Description

Bubble discharging method, particle capturing device, and particle analyzing device.

The present technology relates to a bubble discharging method, a particle capturing device, and a particle analyzing device.

▽Attention is focused on the technology for analyzing particles such as single cells. In the technology of analyzing particles such as single cells, one cell is captured in each of a large number of microwells arranged on a plane, and the morphology of each cell is individually observed to characterize each cell. And/or the reaction of each cell with the reagent can be analyzed using, for example, fluorescence as an index.

As a commercially available device used in the technique of analyzing particles such as single cells, there is, for example, the As One Cell Picking System (As One Co., Ltd.). In the analysis technique using this device, a cell suspension is applied to a microchamber having a large number of wells each having a size for containing one cell, and one cell is precipitated in each of the wells. Then, one cell in each well is individually collected and/or analyzed. The well is provided on the chip in the microchamber. As the chip, a plurality of types of chips are prepared according to the size of cells. For example, a chip in which wells of φ30 μm are arranged at a pitch of 80 μm in the X and Y directions (about 80,000 wells), and a chip in which wells of φ10 μm are arranged at a pitch of 30 μm in the X and Y directions (about 300,000 wells), etc. Is prepared. The characteristics of individual cells isolated in each well by this device are observed by means such as fluorescence detection. The cells of interest can then be extracted from the wells by a micromanipulator, transferred to 96-well/384-well plates and subjected to more detailed analysis, eg sequencing.

Further, as a technique for capturing one cell in one well, for example, a technique described in Patent Document 1 below can be mentioned. Patent Document 1 below describes a "microfluidic device capable of capturing circulating tumor cells (CTC) contained in a blood sample by a size-selective microcavity array, including a sample supply port, a sample discharge port, and a sample supply port. An upper member in which a micro flow channel that connects the port and the sample discharge port is formed, and an opening window for the size selection microcavity array is provided at a position corresponding to a part of the micro flow channel; and below the opening window of the upper member Holding a microcavity array comprising a size-selective microcavity array having fine through-holes whose CTC capture hole diameter, number of holes, and arrangement are controlled at a position corresponding to A portion; a suction opening window provided at a position corresponding to the lower side of the size selection microcavity array, and a lower member having a suction flow path communicating the suction opening window and the suction port. Microfluidic device characterized in that it is described.

Further, Patent Document 2 provides a microchip inspection system capable of removing air bubbles and performing accurate detection even when air bubbles are generated in the liquid inside the detection portion due to heating of the detection portion or the like. The purpose is described. Then, in Patent Document 2, "To that end, at least a target substance and a reagent that specifically binds to the target substance are included, a reaction between the target substance and the reagent is performed, and a reaction is detected in a detected part. A microchip in which is performed, a microchip accommodating portion capable of accommodating the microchip, and a photodetection portion which is provided corresponding to a detected portion of the microchip accommodated in the microchip accommodating portion and detects a reaction, A bubble removing unit that removes bubbles in the detected portion of the microchip housed in the microchip housing unit, and a control unit that controls the photodetector unit to detect the reaction after operating the bubble removing unit. It has a microchip inspection system”.

JP, 2011-163830, A International Publication No. 2008/096563

Due to the recent increasing interest in the life science field, liquid samples such as flow cytometry are increasingly used. However, when handling a fluid, there are always bubbles mixed in the flow channel, and it is difficult to discharge the bubbles from the flow channel. The present disclosure has paid attention to the fact that the mixed bubbles may affect the original measurement or the like, and in the worst case, a false negative or a false positive may occur, which may adversely affect the diagnosis or the judgment.

However, to the present disclosure, as far as the present disclosure is aware, as a measure against the mixed bubbles, the above-mentioned Patent Document 2 (for example, paragraphs [0089] [0095] and the like) describes the mixed bubbles due to the increase in the saturated gas concentration due to pressurization. It only promotes dissolution in the liquid.
In Patent Document 2, since the idea is to discharge the liquid together with the bubbles dissolved in the liquid by pressurization, it was thought that it is difficult to dissolve the bubbles in the liquid if the bubbles become too large. A larger pressure is required to dissolve the bubbles that have become too large. In this case, the present inventor considered that this pressurization is likely to cause troubles such as damage to the device (for example, damage to the flow path of the microchip or the chamber containing the microchip).
Therefore, the present inventor thought that a new attempt to discharge the bubbles mixed in the fluid is necessary, and a technique for discharging such mixed bubbles will be further required in the future.

Therefore, the main purpose of this technology is to provide a technology for efficiently discharging bubbles existing in the fluid in the chamber.

In the present technology, in a chamber including a microchip that has at least one well or through hole and divides into a first space and a second space,
A pressurizing step of applying a positive pressure to the fluid in the chamber,
A valve opening/closing step of operating a valve for opening/closing a first flow path connected to the first space and/or a second flow path connected to the second space. You can
Further, in the present technology, the microchip may be a particle capturing chip having a particle capturing region including at least one well or through hole.
Further, in the present technology, the first flow path is a flow path connected to a first supply valve to which a fluid is supplied and a first discharge valve to which the fluid is discharged, and/or
The second flow path may be a flow path connected to a second supply valve for supplying a fluid and a second discharge valve for discharging the fluid.
In the present technology, the valve opening/closing step may include a step of opening/closing the supply valve and/or the discharge valve after applying a positive pressure to the fluid in the chamber to discharge bubbles from the chamber. ..

In the present technology, the pressurizing step and the valve opening/closing step may be repeated.
The present technology may further include a bubble analysis step of analyzing bubbles in the chamber based on information obtained by imaging the bubbles in the chamber.
The present technology determines whether or not a bubble in the chamber satisfies a predetermined condition in the bubble analysis step, and when the bubble is determined to meet a predetermined condition, the pressurizing step and the valve opening/closing step are performed. You can go.
The present technology, in the pressurizing step, pressurizes the fluid in the chamber with the supply valve opened and the valves other than the supply valve closed.
In the valve opening/closing step, after the supply valve is closed, any one or more of the closed valves may be intermittently opened.
The present technology, in the pressurizing step, pressurizes the fluid in the chamber with the supply valve opened and the valves other than the supply valve closed.
In the valve opening/closing step, any one or more of the closed valves may be opened.
In the present technology, the particles may be single cells.

Further, as another aspect of the present technology, in a chamber having a microchip that has at least one well or through hole and divides into a first space and a second space,
A pressurizing step of applying a positive pressure to the fluid in the chamber, and a valve for opening and closing the first flow path connected to the first space and/or the second flow path connected to the second space. And a pressure control unit for performing a valve opening/closing step to be operated, and controlling so as to discharge bubbles in the chamber,
It is possible to provide a particle capturing device that captures particles in the well or the through hole.
In the present technology, the microchip may be configured such that the upper part of the first space becomes higher in the discharging direction.
In the present technology, the microchip may be configured such that the cross-sectional area in the width direction increases in the discharging direction.

As another aspect of the present technology, in a chamber including a microchip that has at least one well or through hole and divides into a first space and a second space,
A pressurizing step of applying a positive pressure to the fluid in the chamber, and a valve for opening and closing the first flow path connected to the first space and/or the second flow path connected to the second space. A pressure control unit that performs a valve opening/closing step to be operated and controls to discharge bubbles in the chamber,
An observation unit that images a bubble in the chamber and/or particles captured in the well or the through hole with a microscope, and
It is possible to provide a particle analysis device including an analysis unit that performs analysis on the bubbles and/or the trapped particles based on the information acquired from the observation unit.

It is a figure which shows an example of the chamber used for the bubble discharge method which concerns on this technique. It is a schematic diagram which shows the bubble discharge system and bubble discharge method which concern on this technique. It is a schematic diagram which shows an example of the bubble discharge method which concerns on this technique. It is a schematic diagram which shows an example of the bubble discharge method which concerns on this technique. It is a figure which shows the modification of the microchip used for the bubble discharge method which concerns on this technique. It is a figure showing an example of a well or a through hole in a well/through hole region provided in a chamber according to the present technology. It is a figure showing a conceptual diagram of a particle capture device or a particle analysis device concerning this art. It is a figure showing a modification of a chamber concerning this art. It is a block diagram of an example of a device of this art. It is a schematic diagram which shows an example of the well provided in the chamber for particle capture of this technique.

Hereinafter, a suitable mode for carrying out the present technology will be described with reference to the drawings.
The embodiment described below shows an example of a typical embodiment of the present technology, and the scope of the present technology is not narrowly construed by this. The description will be given in the following order. In the drawings, the same or similar elements or members will be denoted by the same reference symbols, and redundant description will be appropriately omitted. Further, the effect produced by the present technology is not necessarily limited to the effect described for each item, and may be any effect described in the present specification.

1. Bubble Discharge Method According to Present Technology <1-1. Bubble Ejection Method of First Embodiment of Present Technology>
<1-2. Bubble Discharge Method of Second Embodiment of Present Technology>
<1-3. Bubble Ejection Method of Third Embodiment of Present Technology>
<1-4. Other embodiments using the bubble discharging method of the present technology>
<1-4(A) Example 1 of particle capturing method and particle analyzing method of the present technology>
<1-4 (B) Example 2 of particle capturing method and particle analyzing method of the present technology>
<1-5. Examples 1 to 3 in the bubble discharging method of the present technology>
2. Device according to the present technology <2-1. Device Having Chamber and Pressure Control Unit According to Present Technology>
<2-2. Chamber and Microchip According to Present Technology>
<2-3. Particle capture device according to the present technology>
<2-4. Particle analysis device according to the present technology>
<2-5. Example 1 of device according to the present technology>
<2-6. Example 2 of device according to the present technology>

1. Bubble Ejection Method According to Present Technology The present technology provides a bubble ejection method in a chamber that includes at least one well or a through hole and includes a microchip that divides into a first space and a second space (for example, (See Figures 1-5). The bubble discharging method comprises a pressurizing step of applying a positive pressure to the fluid in the chamber, and a first flow path connected to the first space and/or a second flow path connected to the second space. And a valve opening/closing step of operating a valve for opening/closing. Thereby, the bubbles existing in the fluid in the chamber can be efficiently discharged.

In the present technology, the chamber is a structure provided with a space for moving the fluid. The fluid is not particularly limited and may be liquid or gas. Further, the fluid may contain particles, and details of the particles will be described later. The microchip may be a microchip for capturing particles, and the microchip preferably has a particle capturing region including at least one well or through hole.
Further, the well, the through hole, the first space and the second space, the space such as the first channel and the second channel, which may be provided in the chamber of the present technology, may be configured to allow the fluid to move appropriately. However, these configurations are not particularly limited.

The chamber of the present technology preferably has at least one well or through hole and is configured to be divided into a first space and a second space. In the present technology, the first space may be a channel through which a fluid can move appropriately or a portion including the channel. In addition, in the present technology, the second space may be a flow path or a portion including the flow path through which the fluid can move appropriately.

The chamber of the present technology preferably has a region including at least one well or one through hole (hereinafter, also referred to as “well/through hole region”). The well or the through hole can communicate with the first space and/or the second space. The well or the through hole may be provided with a hole for communicating with the first space and/or the second space, and the well or the through hole and the first space and/or the second space via the hole. Can be communicated. Although details such as the shape of the well and the through hole will be described later, in the well or the through hole, the opening through which the particles flow may be either upward or downward, but when the opening is upward, in the sedimentation direction such as its own weight. It is preferable from the viewpoint of capturing particles. The well/through hole region can also function as a particle capturing region including at least one well or through hole.

Also, the chamber of the present technology can adopt a configuration including a microchip having at least one well or through hole. Further, the present technology can also adopt a configuration of a microchip that has at least one well or a through hole and divides it into a first space and a second space, and can configure a chamber including the microchip. It is also possible to adopt.

The bubble discharging method of the present technology is a method of performing at least a pressurizing step and a valve opening/closing step in the chamber. It is more preferable that the present technology further perform a bubble analysis step of analyzing bubbles in the chamber. In the bubble discharging method, it is preferable to grasp the condition of the bubbles in the chamber and appropriately process the bubbles in the bubble analyzing step from the viewpoint of efficiently discharging the bubbles.

The bubble discharging method according to an embodiment of the present technology may include a configuration having a first valve and a second valve in each of a first flow path and a second flow path in the chamber, and a pressure control unit for controlling the pressure in the chamber. It is preferable to carry out with an apparatus having such a structure. The number of each of the first valve and the second valve may be singular or plural. The number of each of the first valve and the second valve is preferably at least two or more from the viewpoint of supplying and discharging.

The first flow path of the present technology is preferably a flow path connected to a first supply valve for supplying a fluid and a first discharge valve for discharging a fluid. In addition, the first flow path of the present technology is configured to be connected to the first space and to be connected to a first supply valve to which a fluid is supplied and a first discharge valve to which the fluid is discharged. Is preferred. It is preferable to have a plurality of first flow paths, and it is more preferable to have one on the first supply valve side and one on the first discharge valve side. It is preferable that there is a first space between the first flow path on the first supply valve side and the first flow path on the first discharge valve side, and the first space is connected to both first flow paths. Is. By opening and closing the first supply valve and the first discharge valve, it is possible to easily control the fluid such as the flow velocity and the fluid pressure in the chamber (mainly in the first space).

The second flow path of the present technology is preferably a flow path connected to a second supply valve for supplying a fluid and a second discharge valve for discharging a fluid. In addition, the second flow path of the present technology is configured to be connected to the second space and to be connected to a second supply valve to which a fluid is supplied and a second discharge valve to which the fluid is discharged. Is preferred. It is preferable to have a plurality of second flow paths, and it is more preferable to have one on the second supply valve side and one on the second discharge valve side. There is a second space between the second flow path on the second supply valve side and the second flow path on the second discharge valve side, and the second space is connected to both second flow paths. The configuration is suitable. By opening and closing the second supply valve and the second discharge valve, it is possible to easily control the fluid such as the flow velocity and the fluid pressure in the chamber (mainly in the second space).

Further, the chamber used in the bubble discharging method of the present technology has a region having a well or a through hole between the first space to which the first channel is connected and the second space to which the second channel is connected. It is preferable to adopt a configuration provided with. It is preferable that the first space and the second space are arranged so as to sandwich the well/through hole region like a sandwich structure, and the fluid in the first space or the second space is It is possible to move to the other second space or the first space via the hole area.
The bubble discharging method of the present technology can remove the mixed bubbles more efficiently by controlling the pressure in the chamber having such a configuration.

Note that a fluid such as a buffer solution is allowed to flow into the chamber before the pressurizing step and the valve opening/closing step in the present technology. By flowing the fluid into the chamber, the mixed bubbles can be discharged to some extent, but in reality, the bubbles often remain in the chamber, and it is difficult to completely discharge the bubbles. By performing the bubble discharging method of the present technology, it is possible to efficiently discharge the bubbles mixed in such a chamber. Further, by performing the bubble discharging method of the present technology, bubbles are discharged from the chamber, and the inside of the chamber such as the well/through hole region, the first space or the second space is filled with a fluid in a state without bubbles. Can be satisfied. According to the present technology, it is possible to perform particle trapping, particle analysis, and the like with a fluid in a state where there are less bubbles, and thus it is possible to improve the accuracy of these.

In the present technology, the first space and the second space are arranged in the chamber, but air bubbles mixed in the first space or the second space may exist.
When bubbles are present in the first space in the chamber, it is preferable to discharge the bubbles using the first valve. More specifically, for bubbles existing in the first space in the chamber, it is more preferable from the viewpoint of efficiency that the bubbles are discharged using the first supply valve and the first discharge valve.
Further, when bubbles are present in the second space in the chamber, it is preferable to discharge the bubbles using the second valve. More specifically, for bubbles existing in the second space in the chamber, it is more preferable from the viewpoint of efficiency that the bubbles are discharged using the second supply valve and the second discharge valve.
In the present technology, the first space may be arranged on the upper side, the first space may be the upper flow path and the second space may be the lower flow path, and conversely, the second space may be arranged on the upper space, Can be used as the lower channel and the second space can be used as the upper channel.

The examples 1 to 3 of the basic process including the pressurizing process and the valve opening/closing process in the present technology will be described below (see, for example, FIGS. 3 to 5), but the present technology is not limited thereto. Further, in the present technology, the supply side is a side for supplying a fluid, more specifically, a valve supply side. Further, in the present technology, the discharge side is the side that discharges the fluid, and more specifically, the valve discharge side.

Example 1 of basic process (see, for example, Fig. 3): Open one valve with all the inlet and outlet (supply side and discharge side) valves closed. A positive pressure is applied to the fluid from the open valve to contract the bubbles in the chamber and then the valve is closed. Then, by opening one discharge valve, the bubbles expand, and the bubbles move a certain distance and stop. Then, by intermittently opening the discharge valve, the bubble repeatedly expands and contracts, and the expansion and contraction causes the bubble to move a certain distance and stop. By repeating this, the bubbles are moved to the outlet on the discharge side. For example, as a more specific example, when the discharge valve is opened, the expanding bubble expands and moves toward the discharging side by a certain distance to stop; when the discharge valve is closed, the expanding bubble stops. When the discharge valve is opened again, the bubbles expand and move to the discharge side again and stop. At this time, it is preferable that the supply valve and the discharge valve used are connected to the same space via a flow path or the like. As described above, it is preferable to perform the pressurizing step of applying a positive pressure and the valve opening/closing step of intermittently opening/closing the valve, and it is more preferable to repeat the pressurizing step and the valve opening/closing step. Thereby, the bubbles can be discharged from the chamber.

Example 2 of basic process (see, for example, FIG. 4): Close all inlet/outlet (supply side and discharge side) valves, and then open one valve. In the state where positive pressure is applied to the fluid from the opened valve to contract the bubbles, one discharge valve is opened without closing this valve, and the bubbles are discharged from the outlet on the discharge side in good success. At this time, it is preferable that the supply valve and the discharge valve used are connected to the same space via a flow path or the like. Thus, it is preferable to perform the pressurizing step of applying a positive pressure and the valve opening/closing step of opening the valve in the pressurized state. Thereby, the bubbles can be discharged from the chamber.

Example 3 of basic process (see, for example, FIG. 5): By configuring the height of the upper flow path to increase toward the outlet of the first discharge valve, bubbles can be guided to the outlet. By utilizing the characteristic that bubbles easily move in the direction opposite to their own weight, the upper flow path is provided with a slope that increases toward the outlet. The upper flow path refers to the upper space of either the first space or the second space. By utilizing this structural characteristic, bubbles can be discharged from the chamber.

In the present technology, by appropriately combining Examples 1 to 3 of the basic process, for example, by adopting the combination of Examples 1 and 2 of the basic process, it is possible to perform the exhaust treatment of the bubbles in the chamber with high probability. ..

The bubble discharging method of the present technology will be described in more detail below.
The pressurizing step in the present technology is to apply a positive pressure to the fluid in the chamber.
By applying a positive pressure to the fluid in the chamber, the bubbles in the fluid in the chamber may contract or dissolve in the fluid. For example, the bubbles contracted by the pressurizing process are less likely to come into contact with the inner wall surface or the like when moving the fluid, so that the bubbles easily move along with the flow in the fluid. Further, for example, when the air bubbles that have come into contact with the inner wall surface and the like are contracted by the pressurizing step, the contracted bubbles are separated from the inner wall surface, or the contact surface of the contracted bubbles becomes small, and the contracted bubbles become fluid. It becomes easy to move along the flow.

When positive pressure is applied in the pressurizing step, the side on which the positive pressure is applied may be either the fluid supply side or the fluid discharge side and is not particularly limited. More preferably, when the positive pressure is applied, it is preferable that the fluid is supplied from the side that supplies the fluid (more specifically, the valve supply side), and the side that discharges the fluid when the positive pressure is applied (more specifically This is because it is possible to move bubbles to the valve discharge side).

The means for applying the positive pressure is not particularly limited, and examples thereof include a device capable of adjusting the flow rate and the fluid pressure of the fluid (for example, a pump). The means for applying the positive pressure may be configured separately from the valve opening/closing means, or may be configured to use a pump, a valve or the like in common with the valve opening/closing means.

Further, it is preferable that the means for applying the positive pressure can control the pressurization of the fluid by the pressure control unit. Further, a flow path for applying a positive pressure may be newly provided in the chamber in addition to the first flow path and the second flow path, and may be connected to the first space or the second space. In addition, the first flow path or the second flow path may be used as the flow path for applying the positive pressure, which can simplify the configuration in the chamber.

In the valve opening/closing step of the present technology, for operating a valve for opening/closing a first flow path connected to the first space, and/or for opening/closing a second flow path connected to the second space. It is preferable to operate the valve. By opening the valve after the pressurizing step, the bubbles in the chamber move to the open side. The valve to be opened is preferably on the side that discharges the fluid (for example, the valve discharge side), and the bubbles can be moved to the side that discharges the fluid when the valve is opened. Further, since the bubbles float in the fluid, it is preferable to open the discharge valve connected to the upper space (specifically, the first space or the second space). The bubbles may be sucked and urged to be discharged by a suction means such as a pump connected to the valve on the side for discharging the fluid.

Furthermore, by opening and closing the open valve and then repeating opening and closing intermittently, the bubbles can be moved to the discharge side by expansion and contraction of the bubbles. The above-described pressurizing step may be performed again between the intermittent opening/closing operations to bring the inside of the chamber (particularly the first space or the second section) into a pressurized state. It is preferable since the distance over which the bubbles move can be increased by pressurization and the bubbles can be efficiently discharged.

It is preferable that opening/closing of a valve in the chamber (for example, an opening/closing valve, a flow rate adjusting valve, a flow pressure adjusting valve, etc. or a combination of two or more kinds thereof) can be controlled by a pressure control unit. Thereby, the valve for opening and closing the first flow path and/or the second flow path in the chamber can be operated. By the opening and closing, it is possible to control the fluid (for example, the flow rate or the fluid pressure) that moves in the first space and the second space that are connected to the first flow path and the second flow path, respectively, and to prevent bubbles in the chamber It can be discharged more efficiently.

In the valve opening/closing step in the present technology, it is preferable to appropriately open/close the supply valve and/or the discharge valve after applying a positive pressure to the fluid in the chamber. As a result, the bubbles can be moved by expansion and contraction in the chamber, and the bubbles can be efficiently discharged from the chamber.

Further, it is preferable that the present technology repeats the pressurizing step and the valve opening/closing step. The pressurizing step can shrink the bubbles in the chamber or dissolve the bubbles in the fluid, and the subsequent valve opening/closing step can move the bubbles existing in the chamber to the discharge side. Further, by repeating the pressurizing step and the valve opening/closing step, the moving distance of the bubbles/1 turn is summed, and the bubbles can be efficiently moved to the discharge side. It is preferable to repeat this until the bubbles in the chamber are exhausted. When determining the degree of repetition, it is desirable to perform the particle analysis steps in parallel and observe the movement state of bubbles in the chamber at the observation unit.
By repeating the pressurizing step and the valve opening/closing step, the fluid in the chamber (for example, the flow velocity and the fluid pressure) can be controlled, and the bubbles in the chamber can be efficiently discharged by the control.

-In addition to the pressurization process and the valve opening/closing process, it is preferable that the present technology further perform a bubble analysis process. In the bubble analysis step, the bubbles in the chamber can be discharged better and efficiently. In the bubble discharging method of the present technology, the order and arrangement of the bubble analyzing steps are not particularly limited, and the bubble analyzing steps may be performed at the same time as the pressurizing step or the valve opening/closing step or at another time. Is. It is preferable to perform the bubble analysis step in parallel with the pressurizing step and the valve opening/closing step because it is easy to track the bubble state and determine the situation in which the pressurizing step and the valve opening/closing step are performed.

In the bubble analysis step of the present technology, the bubbles in the chamber are analyzed based on the information obtained by imaging the bubbles in the chamber. Regarding the analysis of bubbles in the chamber, it is possible to make a determination based on the image acquisition information from the observation unit and the analysis unit. In the bubble analysis step, if bubbles are present in the chamber based on the analysis result, pressure control is performed so that the pressurizing step and the valve opening/closing step are appropriately performed until the bubbles are not present in the chamber. It is preferable to instruct a department or the like. Further, by performing the bubble analysis step, the bubbles existing in the fluid in the chamber can be discharged more reliably and efficiently, which can further improve the accuracy of particle trapping, particle analysis and the like.

Further, in the bubble analysis step in the present technology, it is preferable to determine whether or not the bubbles in the chamber meet a predetermined condition. In the bubble analysis step, it is preferable to instruct to perform the pressurizing step and the valve opening/closing step when it is determined that the bubble corresponds to a predetermined condition. The determination unit may be an analysis unit, a pressure control unit, an observation unit, or a central control unit that controls them, and is not particularly limited. Further, when the predetermined condition is stored, these units can be used, but are not particularly limited, and may be stored in an external or internal storage unit. Thereby, the bubbles in the chamber can be efficiently discharged by the control.
The predetermined condition may be set appropriately, or the condition may be set or reset by accumulating bubble information and the like in an artificial intelligence (AI) system.

In the present technology, the size and the number of bubbles when it is determined that there are bubbles in the chamber are not particularly limited and can be appropriately handled. The size of the bubble when it is determined that the bubble is present in the chamber can be appropriately set in advance in each unit such as the storage unit and the analysis unit.
The number of bubbles at the time of the determination is not particularly limited, but preferably, when a single or a plurality of bubbles that do not dissolve in the fluid when pressurized are present in the chamber, such as the number and location of bubbles. It is preferable to output the state of bubbles to the pressure control unit from the viewpoint of efficiency.
The size of the bubble at the time of the determination is not particularly limited, but it is preferable to perform the present technology when the size of the bubble before applying the positive pressure is preferably Φ50 μm or more, and more preferably Φ20 μm or more. When the size of bubbles in the chamber is equal to or smaller than a certain value, it is preferable from the viewpoint of efficiency that positive pressure is applied to the fluid in the chamber to dissolve the bubbles in the fluid to discharge the bubbles.
In addition, when it is determined that the size of the bubbles after applying the positive pressure is larger than a predetermined size, the pressure control is performed such that a step (for example, the second embodiment) in which the bubbles are discharged all at once is adopted. It is preferable to output the data to a unit from the viewpoint of efficiency. When the size of the bubbles when a positive pressure is applied is preferably Φ200 μm or more, and more preferably Φ500 μm or more, it is preferable to perform the step of discharging the bubbles all at once.

Moreover, examples of means for imaging the bubbles in the chamber include a microscope and an imaging device, which will be described later, but are not particularly limited. The means for imaging the bubbles in the chamber may be an observation unit capable of performing particle analysis, or may be configured to cooperate with an analysis unit for analyzing particles or bubbles, Imaging may be controlled by the observation unit and the analysis unit.
It is preferable that the means for analyzing the bubbles adopts a configuration such as an analyzing unit and an observing unit described later, and the information obtained by imaging the bubbles in the chamber may be analyzed.
The means for analyzing bubbles of the present technology is configured to obtain bubble analysis information from other parts, analyze the state of bubbles in the chamber, and examine a method for efficiently discharging bubbles in the chamber. Is preferred.

In the bubble analysis process of the present technology, it is also possible to instruct to set and execute the bubble discharge condition. As the bubble discharging conditions at this time, for example, in addition to the above, selection of each bubble discharging procedure and each step (for example, the first to third embodiments, etc.) in the present technology, selection of a valve to be used, etc. are appropriately set. However, the present technology is not limited to this.
As an example, in the bubble analysis, when it is determined that the bubbles are equal to or less than the predetermined condition, the pressure control unit or the like can instruct to select and execute the bubble discharging method of the first embodiment. Further, in the bubble analysis, the pressure control unit or the like can instruct to select and execute repeating the bubble discharging method of the first embodiment until a condition for determining that there is no bubble is reached.
Further, in the bubble analysis, when it is determined that there are more than a predetermined number of bubbles, the pressure control unit or the like can instruct to select and execute the bubble discharging method of the second embodiment. After that, in the bubble analysis step, when it is determined that the predetermined conditions are not reached, the pressure control unit or the like can instruct the bubble discharging method of the first embodiment to switch.

It should be noted that the bubble discharging method of the present technology is stored as a program in a hardware resource including a control unit including a CPU of various devices, a storage medium (USB memory, HDD, CD, network server, etc.), and It can also be realized. Further, the present technology may be a program for causing a computer to function as the bubble discharging method of the present technology.

According to the present technology, bubbles existing in the fluid in the chamber can be efficiently discharged. This makes it possible to improve the accuracy and efficiency of particle analysis and particle trapping in an apparatus or system for particle analysis, particle trapping, particle analysis, etc. using a flow channel system. For example, in the case of the conventional technique, it is not possible to fractionally capture particles such as cells in a region in which bubbles are mixed, but since the present technique can discharge the mixed bubbles, the number of particles such as cells that can be fractionally captured increases. For example, when counting particles such as cells from the shape by image analysis such as imaging, false positive results will occur if air bubbles of the same size as particles such as cells are mixed. By being able to discharge the generated bubbles, such a thing can be reduced, prevented or avoided. For example, in the case of capturing particles such as cells, in a particle imaging image of cells after capturing particles such as cells, compared to the case where bubbles are mixed, the mixed bubbles can be discharged by the present technology. Better contrast is obtained.
Note that the effects produced by the present technology are not necessarily limited to the effects described here, and may be any effects described in the present specification.

<1-1. Bubble Ejection Method of First Embodiment of Present Technology>
A bubble discharging method according to the first embodiment of the present technology will be described below (see, for example, FIGS. 2 and 3 ).
The start of the bubble discharging method of the present technology is not particularly limited as long as the fluid exists in the chamber. For example, any one of the valves 51, 52, 61, 62 may be opened to allow the fluid to flow into the chamber, or to discharge the bubbles in the present technology from the state where the fluid has already flowed into the chamber. Start. Even if bubbles cannot be confirmed in the chamber, it is preferable to perform the bubble discharging method of the present technology at least once. This makes it possible to discharge bubbles that have been overlooked during observation, and improve the accuracy of subsequent particle capture and particle analysis.

In the first embodiment of the present technology, it is preferable to close all the valves 51, 52, 61, 62 in the presence of fluid in the chamber (see, for example, FIG. 3A). Then, in order to apply a positive pressure, the supply valve is opened, and with the valves other than this supply valve closed, a positive pressure is applied to the fluid in the chamber (see FIG. 3B: pressurizing step). By applying a positive pressure, the bubbles 30 in the chamber shrink and become smaller or dissolve in the fluid.
The supply valve for applying the positive pressure is preferably a supply valve connected to the space where the bubbles 30 are present. For example, when bubbles are present in the first space 3, the first supply valve 51 is preferable, and when bubbles are present in the second space 4, the second supply valve 61 is preferable. Further, when the first space 3 is above, the first supply valve 51 is preferable because bubbles easily float.

Further, in the valve opening/closing step, the supply valve is closed and all the valves 51, 52, 61, 62 are closed. Open any one or more of the closed valves (see, eg, FIG. 3C). By opening the valve, the mixed bubbles move to the opened valve side while expanding.
As the valve to be opened at this time, it is preferable to open a discharge valve connected to the same space (first space or second space) as the supply valve when positive pressure is applied. For example, in the case of the first supply valve 51, the first discharge valve 52 is preferable, and in the case of the second supply valve 61, the second discharge valve 62 is preferable. By opening the discharge valve, the mixed bubbles further move to the discharge side.

By opening and closing the discharge valve, the bubbles mixed in the chamber expand when the discharge valve is opened and contract when the discharge valve is closed, while the bubbles move to the discharge valve side. As the movement of the bubbles at this time, the bubbles move at the time of expansion and stop at the time of contraction.
Further, it is preferable to intermittently open and close the discharge valve, and by intermittently opening and closing, the bubbles can be moved toward the discharge valve while repeatedly moving and stopping due to expansion and contraction of the bubbles. When performing this intermittent opening and closing, the same exhaust valve (eg, the first exhaust valve and the first exhaust valve) may be used, or different exhaust valves (eg, the first exhaust valve and the second exhaust valve) may be used. Good. More preferably, the same discharge valve is used to open and close intermittently.
As shown in FIG. 3, the fluid moves to the first discharge valve 52 side by the pressurizing process and the opening/closing process as described above, and moves to the second discharge valve 62 side via the well/through hole region 9. Can also move.
By such a pressurizing step and a valve opening/closing step, it is possible to more efficiently discharge bubbles from the inside of the chamber.

In the valve opening/closing step, it is preferable to repeatedly open/close the supply valve for applying a positive pressure and then intermittently open/close the discharge valve. More specifically, for example, the supply valve is opened to apply positive pressure to the fluid in the chamber to contract the bubbles, and then the supply valve is closed, and then the discharge valve is opened and closed intermittently to expand and contract the bubbles. Repeat to move to the discharge valve side. Thereby, the bubbles in the chamber can be moved more efficiently to the discharge side. Furthermore, the transfer of bubbles to the discharge side may be further promoted by applying a negative pressure to the discharge valve side by suction or the like. By intermittently opening and closing the valve in this way, it is possible to more efficiently discharge bubbles from the chamber.

In addition, in the bubble discharging method according to the first embodiment of the present technology, when bubbles exist in the first space 3 or the first flow path 5, the first valve (the first valve connected to the first space 3 and the first flow path 5 ( Bubbles are preferably discharged using the first supply valve 51 and the first discharge valve 52). When bubbles are present in the second space 4 or the second flow path 6, the second valve (second supply valve 61, second discharge valve) connected to the second space 4 and the second flow path 6 is provided. 62) is preferably used to discharge the bubbles.
Since bubbles tend to rise in the direction opposite to gravity in the fluid, it is preferable to discharge the bubbles using a valve connected to the space and the flow path above. For example, when the first space is the upper surface and the second space is the lower surface, it is preferable to discharge the bubbles by using the first valve connected to the first space and the first flow path. The method of the first embodiment is effective for small bubbles existing in the chamber.

Furthermore, the state of bubbles in the chamber may be observed by imaging. By observing the bubbles, the bubbles of the present technology can be efficiently discharged until the bubbles in the chamber are discharged. Further, by confirming that no bubbles are present in the chamber by observing bubbles, it is possible to improve efficiency and accuracy of trapping and analysis in, for example, the particle trapping step and the particle analysis step.
In addition, it is possible to determine whether the bubbles in the chamber are present in the first space and the first flow path, or in the space such as the second space and the second flow path by observing the bubbles. For example, as an example, it is determined which of the first space and the first flow path, the second space and the second flow path in the chamber, based on the location where the bubble is focused due to the focus function of the observation unit. You can also Further, it is preferable from the viewpoint of efficiently discharging the bubbles based on the result of observing the bubbles. It is preferable to further perform a bubble analysis step of observing bubbles in the chamber and determining the bubble state.

As described above, according to the bubble discharging method of the first embodiment of the present technology, the bubbles existing in the fluid in the chamber can be efficiently discharged. Furthermore, in the case of the bubble discharging method of the first embodiment of the present technology, consumption of fluid such as a buffer solution can be relatively suppressed.

<1-2. Bubble Discharge Method of Second Embodiment of Present Technology>
A bubble discharging method according to the second embodiment of the present technology will be described below (see, for example, FIGS. 2 and 4). The description of the configuration overlapping the configuration of the bubble discharging method of the present technology (first embodiment and the like) described above will be appropriately omitted.
The start of the bubble discharging method of the present technology is not particularly limited as long as the fluid exists in the chamber.

In the second embodiment of the present technology, it is preferable to close all the valves 51, 52, 61, 62 in the presence of fluid in the chamber (see, for example, FIG. 4A). Then, in order to apply a positive pressure, the supply valve is opened, and with the valves other than this supply valve closed, the fluid in the chamber is pressurized (see FIG. 4B: pressurizing step). By applying a positive pressure, the bubbles 30 in the chamber shrink and become smaller or dissolve in the fluid.
The supply valve for applying the positive pressure is preferably a supply valve connected to the space where the bubbles 30 are present. For example, the first supply valve 51 is preferable when the bubbles 30 are present in the first space 3, and the second supply valve 61 is preferable when the bubbles 30 are present in the second space 4. Further, when the first space 3 is above, the bubbles 30 are likely to float, so the first supply valve 51 is preferable.

Further, in the valve opening/closing step, any one or more of the discharge valves are opened with the supply valve being opened when positive pressure is applied. Since the supply valve is in the open state, a positive pressure is applied from the pump or the like into the chamber, and the valve is opened in the state where the positive pressure is applied, so that the air bubbles in the chamber can be instantly removed. The method of the second embodiment can deal with various bubbles, and is more effective in discharging large bubbles or large and small bubbles existing in the chamber all at once. The second embodiment is preferably performed when the size of the bubbles is large from the viewpoint of the usage amount of the liquid such as the buffer solution.
As a valve to be opened at this time, it is preferable to open a discharge valve connected to the same space (first space or second space) as the supply valve when positive pressure is applied, thereby efficiently moving bubbles. You can For example, in the case of the first supply valve 51, the first discharge valve 52 is preferable, and in the case of the second supply valve 61, the second discharge valve 62 is preferable.
As shown in FIG. 4, the fluid moves to the first discharge valve 52 side by the pressurizing process and the opening/closing process as described above, and moves to the second discharge valve 62 side via the well/through hole region 9. Can also move.

In addition, in the bubble discharging method according to the second embodiment of the present technology, when bubbles exist in the first space 3 or the first flow path 5, the first valve connected to the first space 3 and the first flow path 5 ( Bubbles are preferably discharged using the first supply valve 51 and the first discharge valve 52). When bubbles are present in the second space 4 or the second flow path 6, the second valve (second supply valve 61, second discharge valve) connected to the second space 4 and the second flow path 6 is provided. 62) is preferably used to discharge bubbles.
Since bubbles in the fluid tend to rise in the direction opposite to gravity, it is preferable to discharge bubbles using a valve connected to the space and the flow path above. For example, when the first space is the upper surface and the second space is the lower surface, it is preferable to use the first valve connected to the first space and the first flow path to discharge the bubbles.

Further, similarly to the bubble discharging method of the first embodiment of the present technology described above, the state of bubbles in the chamber may be observed by imaging. By observing the bubbles, the method can be efficiently performed until the bubbles in the chamber are exhausted.

Thus, according to the bubble discharging method of the second embodiment of the present technology, the bubbles existing in the fluid in the chamber can be discharged efficiently. Furthermore, in the case of the bubble discharging method according to the second embodiment of the present technology, the bubbles are discharged when the bubbles are large or adhere to the wall surface, or when the bubbles are scattered in the observation surface, that is, when the number of bubbles is large. Difficult air bubbles can be discharged at once.

In order to discharge the bubbles in the chamber, the bubble discharging method according to the first embodiment of the present technology and the bubble discharging method according to the second embodiment may be appropriately combined depending on the state of bubbles, or these methods may be alternately performed. You may

<1-3. Bubble Ejection Method of Third Embodiment of Present Technology>
As a third embodiment, air bubbles are discharged using a microchip (for example, see FIG. 5) configured such that the upper side of the first space becomes higher in the discharging direction. It is preferable to perform the pressurizing step and the valve opening/closing step as the discharge of bubbles, and at this time, it is more preferable to appropriately adopt the bubble discharging method or the second bubble discharging method of the first embodiment of the present technology described above. It is suitable.
FIG. 5 shows an example of a microchip used in the third embodiment of the present technology, but the present technology is not limited to this. As shown in FIG. 5, in a chamber that includes at least one well 2 or through hole and includes a microchip 10 that is divided into a first space 3 and a second space 4, the microchip 10 is located above the first space 3. Is configured to be higher in the discharging direction.
Hereinafter, an example of the bubble discharging method of the present technology using the microchip of the third embodiment of the present technology will be shown, but the method is not limited thereto.

According to the first embodiment or the second embodiment described above, by using a microchip configured such that the upper part of the first space becomes higher in the discharging direction and the cross-sectional area in the width direction becomes larger in the discharging direction. , Pressurizing step and valve opening/closing step. Thereby, the bubbles existing in the fluid in the chamber can be efficiently discharged.

By adopting such a configuration that the cross-sectional area is intentionally increased in the outlet direction, the flow velocity in the chamber can be reduced. On the other hand, when it is desired to keep the flow velocity in the chamber constant, it is possible to adopt a configuration in which the cross-sectional area of the microchip is constant.
For example, make the cross-sectional area constant by narrowing the width along the exit direction by increasing the height along the exit direction of the microchip; increasing the height near the center of the microchip and lowering the left and right sides. Such a trapezoidal shape may be used to make the cross-sectional area constant.

<1-4. Other embodiments using the bubble discharging method of the present technology>
<Particle capture method and particle analysis method of the present technology>
The bubble discharging method of the present technology can be appropriately applied to an apparatus including a chamber and a method of using the apparatus. In addition, since the present technology may include particles in the fluid, it can be used for particle trapping and particle analysis. The bubble discharging method of the present technology can be suitably incorporated into, for example, a particle capturing method or a particle analyzing method using a chamber, but is not limited thereto.

The bubble discharging method of the present technology can be performed as a pretreatment step in a particle capturing method or a particle analyzing method, for example. Further, the bubble discharging method of the present technology can be appropriately performed in parallel with or during the particle capturing method and the particle analyzing method, as needed. Bubbles may be mixed or left in the middle of the steps of the particle capturing method and the particle analyzing method. Therefore, by using the bubble discharging method of the present technology, such bubbles can be discharged.
As described above, the present technology can also provide a particle capturing method including the bubble discharging method of the present technology, or a particle analysis method including the bubble discharging method of the present technology. Further, the present technology can also provide a particle capturing device or a particle analysis device having a configuration capable of executing the bubble discharging method of the present technology.

In the present technology, it is preferable that the microchip is a particle capturing chip having a particle capturing region including at least one well or through hole. Furthermore, it is preferred that the particles are single cells. By using the bubble discharging method of the present technology, bubbles existing in the fluid in the chamber can be discharged efficiently, so that particles (for example, single cells) can be easily captured. In addition, in the present technology, it is preferable to perform a particle analysis step of performing analysis regarding particles.

The “particles” are preferably those that are required to be captured or analyzed one by one, for example. Examples of the particles include, but are not limited to, cells, microorganisms, biologically-derived solid components, biological microparticles such as liposomes, and synthetic particles such as latex particles, gel particles, and industrial particles. Not done. The cells may include animal cells and plant cells. Animal cells can include, for example, tumor cells and blood cells. The microorganism may include bacteria such as Escherichia coli and fungi such as yeast. Examples of the solid component derived from the living body include solid matter crystals produced in the living body. The synthetic particles may be particles made of, for example, an organic or inorganic polymer material or a metal. The organic polymer material may include polystyrene, styrene/divinylbenzene, polymethylmethacrylate and the like. The inorganic polymer material may include glass, silica, magnetic material and the like. The metal may include colloidal gold and aluminum. Further, in the present technology, the particles may be a combination of a plurality of particles such as two or three.

The particle capturing method and the particle analyzing method of the present technology are not particularly limited as long as they are methods that can be executed by various particle capturing apparatuses and particle analyzing apparatuses that can use the chamber of the present technology described above.
Examples 1 and 2 of the particle capturing method and the particle analyzing method are shown below, but the present invention is not limited to these examples, and the bubble discharging method of the present technology can be used for general particle capturing methods and particle analyzing methods. Is.

<1-4(A) Example 1 of particle capturing method and particle analyzing method of the present technology>
An example of the particle capturing method and the particle analyzing method used in the present technology will be described with reference to the apparatus illustrated in FIG. 7, but the method and apparatus of the present technology are not limited to this example.
For example, as an example of the particle trapping method of the present technology, a particle trapping step, a step of removing untrapped particles, an analysis step of trapped particles, a step of acquiring desired particles from trapped particles, and trapping An example in which a recovery step of the other particles thus obtained is performed can be given, but the present technology is not limited thereto.

In step S100, the above-described bubble discharging method of the present technology (more preferably, the first to third embodiments) can be performed as a pretreatment of the particle capturing method or the particle analyzing method. Thereby, the bubbles existing in the fluid in the chamber can be efficiently discharged. Then, the particle capture and the particle analysis after step S101 are started.

In step S101, the particle capturing method used in the present technology is started. Prior to the start of the particle capturing method, the fluid containing the particles S is placed in the container 70.

In step S102, a particle capturing step is performed. Prior to the supply or suction of the particles, the particle supply valve 72 connected to the particle flow passage 71 provided between the first flow passage 5 and the container 70 may be closed. The opening/closing of the particle supply valve 72 can be appropriately adjusted according to the supply status of particles to the first space 3. The particle supply valve 72 can control the supply of particles. In the particle capturing step, the particle supply valve 72 connected to the particle flow path 71 is opened, and supply or suction by the pump (P in the figure) on the first supply valve 51 side or the first discharge valve 52 side is started. To be done. When started, the fluid containing the particles S enters the first flow path 5 from the container 70 via the first supply valve 51 by the supply or suction. By controlling the opening and closing of the first supply valve 51 and the first discharge valve 52, the flow rate and flow velocity of the fluid are controlled. The fluid containing the particles S enters the first space 3 of the particle capturing chamber 100 through the first flow path 5. Further, by continuing the supply or suction of the fluid by the pump, the particles S enter the well 2 by their own weight or sedimentation. The particles S that have entered the well 2 collide with the entrance of the hole 8, whereupon the particles S stop moving. In this way particles are trapped within the well 2.

In step S103, a step of removing particles S not captured by the well is performed. In the removal step, the particles S that have not been captured by the wells can be discharged from the particle capturing chamber 100. The particles S not captured in the well can be discharged through the first flow path 5 and the first discharge valve 52 by sucking with the pump connected to the first discharge valve 52.

In step S104, an analysis process of particles trapped in the well is performed. In the analysis step, for example, observation with the inverted microscope 80 can be performed. Further, in the analysis step, analysis may be performed by an analysis device other than the inverted microscope. In the analysis step, the fluorescence emitted by each particle can be analyzed by, for example, a photodetector.

In step S105, a step of obtaining desired particles from the captured particles is performed. In the acquisition step, first, desired particles are selected as a result of the analysis in step S104. For example, particles having a desired morphology or particles that emit a desired fluorescence can be selected. The selected particles are then acquired by a single particle acquisition device such as a micromanipulator.

In step S106, another trapped particle, that is, a particle not selected in step S105 is collected. First, the first discharge valve 52 is opened, and then a pressure (for example, positive pressure) is applied by the pump connected to the first discharge valve 52 so that the particles S come out of the well 2. The particles S emitted from the well 2 pass through the first flow path 5 on the first discharge valve 52 side and are collected in a container (not shown).

In step S107, the particle capturing method of the present technology is completed.

In the above flow, it is possible to observe particles one by one. It is also possible to acquire only one target particle. Further, other particles captured in the wells and particles not captured in the wells can be collected, and these particles can be used for other tests. Further, in the above flow, a particle capturing chamber having a through hole may be used instead of the well.

Note that, in Step S101 to Step S107 described above, when bubbles are present in the chamber, the bubble discharging method of the present technology can be appropriately performed. Thereby, air bubbles can be appropriately discharged from the chamber. Preferably, by performing immediately before the particle capturing step of step S102, bubbles can be discharged and the number of particles that can be fractionally captured can be further increased. Further, preferably, by performing immediately before the particle analysis step of step S104, it is possible to discharge bubbles, and improve the contrast of the imaging image of particles and the like and the accuracy of particle counting.

<1-4 (B) Example 2 of particle capturing method and particle analyzing method of the present technology>
Although an example of the particle capturing method and the particle analyzing method used in the present technology will be described with reference to the apparatus illustrated in FIG. 8, the method and apparatus of the present technology are not limited to this example. The bubble discharging method of the present technology can be applied even in the case where the chamber is directed toward the opening for trapping particles downward and trapped by suction as shown in FIG. The same configurations as those in the above-mentioned example 1 are omitted as appropriate.

In step S200, the above-described bubble discharging method of the present technology (more preferably, the first to third embodiments) can be performed as a pretreatment of the particle capturing method or the particle analyzing method. Thereby, the bubbles existing in the fluid in the chamber can be efficiently discharged. Then, the particle capture and the particle analysis after step S201 are started.

In step S201, the particle capturing method used in the present technology is started. A fluid containing particles is placed in a container (not shown) prior to starting the particle capture method.

In step S202, a particle capturing step is performed. Prior to the suction of the particles, the valves 51, 52, 61, 62 may be closed. In the particle capturing step, the first supply valve 51 or the first discharge valve 52 is opened, and suction by the pump (P in the figure) connected to each is started. When started, the suction causes the fluid containing the particles S to enter the second space 4 on the sedimentation side of the particle capturing chamber 100 from the container through the particle flow passage 71. By further continuing the suction, the particles S float in the second space 4 on the sedimentation side and enter the well 2. The particles S that have entered the well 2 collide with the entrance of the hole 8, whereupon the particles S stop moving. In this way, the particles S are trapped in the well 2. In the particle capturing step, the suction is stopped or the suction force is reduced after a predetermined time has elapsed from the start of the suction. This stops the particles from floating in the chamber, and the particles not captured in the well settle on the bottom surface of the chamber.

In step S203, a step of removing particles not captured by the well is performed. In the removal step, particles that have not been captured in the wells can be discharged from the particle capturing chamber 100. For example, by suctioning with a pump connected to the particle channel 71, particles settled on the bottom surface of the chamber are discharged from the chamber 100 through the particle channel 71, and then a container (not shown). ).

In step S204, the step of analyzing the particles captured in the well is performed.
In step S205, a step of obtaining desired particles from the captured particles is performed. The steps S204 and S205 may be the same steps as the steps S104 and S105, and will be omitted.

In step S206, another trapped particle, that is, a particle not selected in step S205, is collected. First, the first supply valve 51 or the first discharge valve 52 is opened and a pump connected to the first supply valve 51 or the first discharge valve 52 applies a pressure (for example, a positive pressure) so that the particles come out of the well. The particles emitted from the well can pass through the particle channel 71 and be collected in a collecting container (not shown).

In step S207, the particle capturing method of the present technology is finished.

In the above flow, it is possible to observe particles one by one. It is also possible to acquire only one target particle. Further, other particles captured in the wells and particles not captured in the wells can be collected, and these particles can be used for other tests. Further, in the above flow, a particle capturing chamber having a through hole may be used instead of the well.

Note that, in Step S201 to Step S207 described above, when bubbles are present in the chamber, the bubble discharging method of the present technology can be appropriately performed. Thereby, air bubbles can be appropriately discharged from the chamber. Preferably, by performing immediately before the particle capturing step of step S202, it is possible to further discharge the bubbles and increase the number of particles that can be fractionally captured. Further, preferably, by performing immediately before the particle analysis step of step S204, it is possible to discharge bubbles to improve the contrast of the imaging image of particles and the like and the accuracy of particle counting.

With respect to the present technology, those skilled in the art will understand that various modifications, combinations, sub-combinations, or alternatives are possible within the scope of the present technology and equivalents thereof, for example, depending on design requirements or other factors. to understand.

<1-5. Examples 1 to 3 in the bubble discharging method of the present technology>
Hereinafter, the present technology will be described in more detail based on Examples and the like. The embodiments and the like described below are examples of typical embodiments and the like of the present technology, and the scope of the present technology is not narrowly construed by this.

[Basic configuration]
As a mode for realizing the present disclosure, as shown in the overall configuration diagram of FIG. 1 and FIG. 2, microwells arrayed in a mesh shape, and valves that can be opened and closed at the entrances and exits of the upper flow path and the lower flow path by sandwiching the well array above and below 51, 52, 61, 62, and the pressure control unit 20 is assumed to be located at the end. Then, the positive and negative pressure control by the pressure control unit 20 and the operation of each valve are performed to discharge the mixed bubbles.
・Microwell array (see Fig. 10)
When the cell diameter is assumed to be φ10 to 20 μm, it is designed with the following specifications, for example.
・Microwell: φ20μm x depth 20μm
-Fine through holes: Slit shape/width 3.2 μm x length 10 μm x depth 15 μm
-Substrate material: plastic resin such as glass/acrylic and polystyrene/rubber such as PDMS-Well pitch: 60 μm each in X and Y directions
・Number of wells: ~7,000
・Microwell area size: X direction_8 mm x Y direction_8 mm
These numbers are not limited to this, and can be optimized in any way depending on the purpose and cell type.

Examples will be described below. When air bubbles are mixed in the upper flow path, air bubbles are mixed in the lower flow path using the first supply valve 51 (upper IN valve) and the first discharge valve 52 (upper OUT valve). In this case, the second supply valve 61 (lower IN valve) and the second discharge valve 62 (lower OUT valve) are used to remove mixed bubbles.

[Example 1]
Example 1 will be described with reference to FIG. When air bubbles are mixed in during priming with a buffer solution or the like (FIG. 3A), the first supply valve 51 (upper IN valve) is opened and a positive pressure is applied up to about +Δ300 hPa. Contracts. In this state, if the first supply valve 51 (upper IN valve) is closed and the first discharge valve 52 (upper OUT valve) is opened, the mixed bubbles slightly move to the outlet side as shown in FIG. 3C. At this time, a negative pressure may be applied to the first discharge valve (upper OUT valve) side to promote the movement to the outlet side. By repeating the operations of (FIG. 3B) and (FIG. 3C), the mixed bubbles 30 can be moved to the outlet. With this method, the consumption of the buffer solution can be relatively suppressed.

[Example 2]
Example 2 will be described with reference to FIG. When air bubbles are mixed in during priming with a buffer solution as in Example 1 (FIG. 4A), the first supply valve 51 (upper IN valve) is opened, and a positive pressure is applied up to about +Δ300 hPa (FIG. 4B). The bubbles 30 thus mixed contract. At this time, when the first discharge valve (upper OUT valve) is opened with the positive pressure applied without closing the first supply valve 51 (upper IN valve), the mixed bubbles 30 as shown in FIG. 3C are generated. Move vigorously to the exit side. At this time, the first discharge valve 52 (upper OUT valve) side may be set to a negative pressure to further promote the movement to the outlet side. According to this method, the mixed bubbles can be instantly removed as compared with the first embodiment.

[Example 3: Application example]
Since air bubbles tend to travel in the direction opposite to their own weight, they are often mixed in the upper channel of the well array. Therefore, as shown in FIG. 5, bubbles are guided to the outlet by raising the upper part (ceiling) of the upper flow path toward the outlet of the first discharge valve 52 (upper OUT valve). Specifically, the height on the side of the first supply valve 51 (upper IN valve) is 0.1 mm and the width is up to 0.7 mm, the height on the side of the first discharge valve 52 (upper OUT valve) is 0.3 mm and the width up to 1. By setting the distance to 5 mm, the bubbles actually move well to the outlet side. By adding the form of the upper flow path and the valve operation, it is possible to discharge the mixed bubbles with higher probability.
In this application example, the cross-sectional area is intentionally increased in the direction toward the outlet, so that the flow velocity is slowed down. However, in order to keep the flow velocity constant, 1) the height is increased, This can be dealt with by making the cross-sectional area constant, such as narrowing the width, 2) increasing the height only in the center and lowering it on the left and right.

In addition, since small bubbles may be dissolved in the liquid just by applying positive pressure, it is possible to determine the size of the bubbles at the same time as determining the presence or absence of bubbles by imaging etc. 1) Only applying positive pressure 2) A system that automatically selects positive pressure application+valve operation can be easily considered.

2. Device according to the present technology <2-1. Device Having Chamber and Pressure Control Unit According to Present Technology>
The device according to the present technology is configured to be capable of executing the above-described bubble discharging method of the present technology, and may be a bubble discharging device for discharging bubbles in the chamber.

The device according to the present technology is preferably a device including a chamber and a pressure control unit. It is preferable that the chamber has at least one well or one through hole and includes a microchip that divides the first space and the second space. The pressure control unit preferably performs a pressurizing step of applying a positive pressure to the fluid in the chamber and a valve opening/closing step, and controls so as to discharge bubbles in the chamber. .. Further, the pressure control unit executes a valve opening/closing step to open a valve for opening/closing the first flow path connected to the first space and/or the second flow path connected to the second space. It is possible to operate.
Further, some or all of the functions performed by the pressure control unit may be arranged outside the device according to the present technology, or may be arranged inside an accessible information processing device (for example, a server). Good. Further, the pressure control unit of the present technology may work in cooperation with another control unit or the central control unit, or may be incorporated in the other control unit or the central control unit.

This technology can be equipped with a pressure control device, a valve, a pump, an observation device, an analysis device, etc., as needed. The observation device can observe the location, size, etc. of bubbles existing in the chamber. Further, the analysis device can appropriately perform analysis such as bubble analysis and particle analysis.

Further, the device of the present technology may be a particle capturing device that captures particles in the well or the through hole, including a bubble discharging device capable of executing the method of the present technology. Further, the device of the present technology may be a particle analysis device for analyzing particles, which includes a bubble discharging device capable of executing the method of the present technology.
Note that with respect to the device according to the present technology and the configuration and steps related to this operation, overlapping with the configurations and steps described above will be appropriately omitted.

The chamber of the device according to the present technology is preferably a structure provided with a space for moving a fluid or a fluid containing particles. Further, the chamber of the present technology can adopt a configuration including a microchip having at least one well or through hole. In addition, the present technology can adopt a configuration of a microchip that has a well or a through hole and is divided into a first space and a second space, and also employs a configuration of a chamber including the microchip. Is also possible.

In the present technology, at least one well or one through hole is provided and is configured to be divided into a first space and a second space (see, for example, FIG. 1 and the like). The present technology preferably has a region including at least one well or one through hole.
Further, in the present technology, the first space and the second space may each be a flow path or a portion including the flow path through which a fluid or a fluid containing particles can appropriately move. Furthermore, in the present technology, the first flow path is connected to the first space and/or the second flow path is connected to the second space.

Further, in the present technology, a valve for opening/closing a first flow path connected to the first space is provided, or a valve for opening/closing a second flow path connected to the second space is provided. Is preferred. In addition, in the present technology, it is preferable to include a valve for opening and closing either or both of the first flow path connected to the first space and the second flow path connected to the second space.
In the bubble discharge of the present technology, the type of valve is not particularly limited and can be appropriately selected by those skilled in the art. As the valve, a commercially available one can be used.
Further, in the bubble discharge of the present technology, the number of valves may be one or plural, but two or more are preferable because it is easy to control the bubble discharge. Then, it is preferable to connect the two valves to the supply side and the discharge side of the flow path, respectively.

In the present technology, the means for settling the particles is not particularly limited, and examples thereof include gravity, centrifugal force, suction, and extrusion. The means for floating the particles is not particularly limited, and examples thereof include suction and extrusion. For example, a structure (for example, a pump or the like) that sucks or pushes out the fluid in the flow path may be provided, and the particles may be moved to the well or the through hole by sucking or pushing.
Further, in the present technology, the suction or the extrusion may be performed by any means known to those skilled in the art, and may be performed by, for example, a pump. A commercially available pump may be used as the pump. The type of pump is not particularly limited and can be appropriately selected by those skilled in the art depending on, for example, the suction force or the pushing force to be applied.

<2-2. Chamber and Microchip According to Present Technology>
A preferred embodiment of the chamber or microchip of the present technology is described below.
In the chamber or microchip of the present technology, holes may be provided in the well (see, for example, FIG. 6). The well may be communicated with the first channel and/or the second channel via the hole. That is, the hole penetrates the well region from the well side to the first flow channel side and/or the second flow channel side. Particles may be transferred into the well by performing suction through the first channel and/or the second channel through the hole. The number of holes provided in each well can be, for example, 1 to 10, in particular 1 to 5, and more particularly 1 to 3. From the standpoint of ease of manufacture, the number of holes provided in each well may be 1 or 2, especially 1.

In the chamber or microchip of the present technology, any shape may be adopted as the shape of the entrance of the hole. In the present technology, the entrance of a hole refers to the opening of the hole in the wall surface of the well in which the hole is provided. The shape of the entrance of the hole can be, for example, a circle, an ellipse, a polygon, such as a triangle, a quadrangle (such as a rectangle, a square, a parallelogram, and a rhombus), a pentagon, or a hexagon (see, for example, FIG. 4 ). In the present technology, the shape of the entrance of the hole may be preferably rectangular, more preferably rectangular or square, and even more preferably rectangular.

In the chamber or the microchip of the present technology, it is desirable that the entrance of the hole can have a size that prevents particles contained in the fluid from passing through the hole and advancing to the other flow path side by suction. For example, the minimum size of the pore entrance is less than the size of the particles.
For example, in the case where the shape of the entrance of the hole is a rectangle, a size smaller than the size of the particles contained in the fluid (for example, the diameter of the particles) is set as the short side or the long side of the rectangle, particularly the short side of the rectangle. Can have. For example, the length of the short side of the rectangle is not particularly limited as long as it is set so as not to hinder suction, and for example, is 0.01 times or more the size of particles contained in the fluid (for example, the diameter of particles). 9 times or less. Such a shape of the holes may allow particles to be trapped while suppressing particle damage.

In the chamber or microchip of the present technology, the shape of the entrance of the hole is preferably rectangular. The length of the long side of the rectangle is preferably 1.2 times or more and 5 times or less the length of the short side of the rectangle. With such a slit shape, damage to particles when the particles are trapped in the well can be suppressed. Such a slit shape is particularly preferable when the particles are cells. Since the entrance of the hole has the slit shape, damage to the cell is suppressed while preventing the cell from passing through the hole. For example, the shape of the entrance of the hole may be a slit shape having a short side of 1 μm to 10 μm, particularly 2 μm to 8 μm, and a long side of 5 μm to 20 μm, particularly 6 μm to 18 μm.

In the chamber or microchip of the present technology, the hole may be preferably provided at the bottom of the well. When the hole is provided at the bottom of the well, the length of the hole is shorter than when the hole is provided on the side surface of the well. As a result, manufacturing may be easier. The bottom of the well may be, for example, a wall on the opposite side of the well entrance from the walls forming the well.
From the viewpoint of workability, the holes are preferably shallower. On the other hand, the holes are preferably deeper from the viewpoint of strength in capturing particles. Therefore, in the present technology, when the hole is provided at the bottom of the well, the depth of the hole (that is, the distance from the well bottom surface to the surface opposite to the particle trapping surface) is preferably 5 to 100 μm, more preferably 6 μm. It can be ˜50 μm, and even more preferably 10-30 μm.
The “particle trapping surface” in the present technology is a surface when particles moving in the flow path enter the well or the through hole. In addition, in the present technology, the surface on which the well is provided or the surface on which the through hole is provided is also referred to as a “particle trapping surface”.

In the chamber or microchip of the present technology, the well may be opened in either direction of the first space or the second space, the direction of the inlet of the well is not particularly limited, and the opening may be formed on the particle capturing surface. It is suitable. For example, when capturing by sedimentation such as particle self-weight or suction, the inlet of the well may face the side opposite to the particle sedimentation side, which allows particles moving in the chamber to move due to self-weight or suction. Can be captured in the well. On the other hand, when the particles are trapped by the force opposite to the sedimentation, for example, the well inlet is preferably directed toward the sedimentation side of the particles, which allows suction to the side opposite to the sedimentation side of the particles. Particles moving in the chamber at can be trapped in the well.

In the chamber or the microchip of the present technology, each well may have a shape capable of capturing one particle (see, for example, FIG. 6 ). For example, well inlets can be, for example, circular, oval, polygonal, such as triangular, quadrangular (such as rectangular, square, parallelogram, and rhombus), pentagonal, and hexagonal. In the present technology, the well inlet refers to the well inlet on the surface on which the well for capturing particles is provided. The shape of the well entrance can be designed, for example, such that particles to be captured can enter the well, but particles that are not to be captured cannot enter the well.
In other embodiments, the well may have a shape such that the well entrance is narrowest and the interior of the well has a larger cross-sectional area. With such a shape, particles that have entered the well can be prevented from leaving the well.
In still other embodiments, the wells may be shaped such that the well entrance is widest and the interior of the well has a smaller cross-sectional area. Such a shape may allow particles to enter the well more easily.

In the chamber or microchip of the present technology, the through hole plays the same role as the hole. All the explanations for the holes also apply for the through holes. For example, the above description regarding the shape and dimensions of the entrance of the hole also applies to the description of the two openings of the through hole (in particular, the opening where particles are trapped among the two openings).
Further, the length of the through hole (that is, the distance between the two openings) may be the same as the thickness of the through hole region, and particularly the thickness of the plate-shaped portion described below.
The shape of the through hole may be, for example, a cylinder, a prism (for example, a triangular prism or a quadrangular prism), a mountain shape, or a shape other than these.
For example, when the shape of the through hole is a quadrangular prism, the shape of the mouth on the particle trapping surface of the through hole may be a rectangle, and the rectangle may be continuous to the opposite surface.
Further, when the shape of the through hole is a mountain shape, the side surface (that is, the inclined surface) of the through hole may be linear or curved (for example, an arc-shaped surface). In this case, the particles may be trapped near the mouth of the through hole, or may be trapped in the middle of the through hole.
Further, as another shape of the through hole, for example, the shape of the mouth of the through hole in the particle trapping surface is a shape that is continuous up to the middle of the through hole, and the cross-sectional area of the through hole gradually decreases from the middle Good. As an example of such a shape, for example, a microneedle shape or the like can be mentioned.

The well region or the through hole region in the chamber or the microchip of the present technology may have at least one surface on which the well or the through hole is provided. In the present technology, the surface on which the well is provided or the surface on which the through hole is provided so that particles can be captured is also referred to as a particle capturing surface.
The particle capturing surface may be a flat surface or a curved surface. From the viewpoint of ease of production, the particle capturing surface is preferably a flat surface. When the particle trapping surface is a plane, the particle trapping surface may be provided so that the plane is perpendicular to the action direction of gravity on the particles, or the plane is 90 degrees with respect to the action direction. The particle trapping surface may be provided to form an angle of less than.

In the chamber or microchip of the present technology, the wells or the through holes can be regularly arranged on the surface for capturing particles. The regular well or through hole arrangement makes it easier to locate the well or through hole in which the particles of interest are trapped. As a result, it becomes possible to more easily take out and/or observe particles captured by, for example, a well or a through hole. For example, the wells or the through holes may be arranged in a row or a plurality of rows on the particle capturing surface at a predetermined interval, or the wells or the through holes may be arranged in a lattice pattern on the particle capturing surface at a predetermined interval. The interval can be appropriately selected by those skilled in the art depending on, for example, the number of particles to be applied and the number of particles to be captured. The spacing may be, for example, 20 μm to 300 μm. For example, when the wells or through holes are arranged in a grid pattern, the wells or through holes may be arranged at the intervals illustrated above in the X direction and the Y direction on the particle capturing surface.

In the chamber or the microchip of the present technology, more preferably, the well region or the through hole region may be arranged so as to divide the inside of the chamber into a first space and a second space. Due to this division, the well/through hole region has a surface in contact with the first space or the second space.
In the present technology, it is preferable that the well region or the through hole region is a particle capturing region, and at this time, the particle capturing region has at least one surface for capturing particles by the partition. More preferably, it is a particle capturing chip having the particle capturing region.
In the chamber or the microchip of the present technology, when the particle capturing region has a well or a through hole, particles can move in the chamber by sedimentation or suction and be captured in the well. The direction of the opening for capturing the particles in the well or the through hole in the region is not particularly limited. The particle trapping region can be provided in the chamber to enable such particle movement and trapping.

In the chamber or the microchip of the present technology, the range of the number of wells or through holes is not particularly limited, and is, for example, 1 to 1,000,000, particularly 10 to 800,000, more particularly 100 to 600,000. Even more particularly can be 1,000 to 500,000.

In the present technology, the first space may be a channel for flowing a fluid or a fluid containing particles or a portion including the channel. Further, in the present technology, the second space may be a channel for flowing a fluid or a fluid containing particles or a portion including the channel.

In the chamber or the microchip of the present technology, a fluid or a fluid containing particles can move in the first space, the second space, the first channel or the second channel. It is preferable that these are configured so that they can be used when capturing particles in the well or in the through holes. The first space is preferably connected to the first flow path. The second space is preferably connected to the second flow path. Thereby, suction can be performed through the first channel and/or the second channel so that the particles are trapped in the well or in the through hole.

In the chamber or the microchip of the present technology, the particle capturing area may be connected to the suction section. The suction can be performed by the suction unit. The suction part can be, for example, a pump known to a person skilled in the art. The pump used in the present technology is preferably a pump capable of finely adjusting the suction force, and more preferably a pump capable of controlling the pressure on the order of several tens Pa near 1 kPa. Such pumps are commercially available, for example, KAL-200 (Hull Strap Co.) can be mentioned.

In the chamber or the microchip of the present technology, the particle capturing region is not only used for capturing the particles in the well or in the through hole, but also for discharging the particles captured in the well from the well or in the through hole. It can also be used in releasing trapped particles from the through hole. For example, when suction is performed by negative pressure, the discharge can be performed by applying positive pressure.

The accuracy and efficiency of cell capture and cell image analysis can be further improved by the combined effect of the chamber or microchip (for example, configuration and structure) of the present technology and the bubble discharging method of the present technology.

<2-3. Particle capture device according to the present technology>
Regarding the configuration and steps related to the particle capturing apparatus and the operation according to the present technology, duplication of the configurations and steps described above will be appropriately omitted. In addition, configurations that overlap with Examples 1 and 2 of the device according to the present technology, which will be described later, are appropriately omitted.

The particle capture device of the present technology is a device that includes a chamber that has at least one well or through hole and that includes a microchip that divides into a first space and a second space. Furthermore, the particle trapping device of the present technology includes a pressurizing step of applying a positive pressure to the fluid in the chamber, a first flow path connected to the first space, and/or a first flow path connected to the second space. The valve opening/closing step of operating a valve for opening/closing the two flow paths is performed to discharge bubbles in the chamber. The particle capture device of the present technology further includes a control unit that controls the pressurizing process and the valve opening/closing process.
Furthermore, the particle capturing device according to the present technology has at least one well or through hole, and is configured to capture particles in the well or through hole in a chamber including a microchip that divides the first space and the second space. The device is configured in. Further, it is preferable to further include an observation unit and an analysis unit described below.
It is preferable that the microchip is a particle capturing chip having a particle capturing region including at least one well or through hole. It is also preferable that the particles are single cells.

In the present technology, bubbles existing in the fluid in the chamber can be efficiently discharged by the pressure control unit. This makes it possible to improve the accuracy and efficiency of capturing particles. For example, in the case of the conventional technique, it is not possible to fractionally capture particles such as cells in a region in which air bubbles are mixed, but since the present technique can discharge the mixed bubbles, the number of particles such as cells that can be fractionally captured increases.
Note that the effects produced by the present technology are not necessarily limited to the effects described here, and may be any effects described in the present specification.

<2-4. Particle analysis device according to the present technology>
Regarding the particle analysis apparatus according to the present technology and the configuration and steps related to this operation, overlapping description with the configuration and steps described above will be appropriately omitted. In addition, configurations that overlap with Examples 1 and 2 of the device according to the present technology, which will be described later, are appropriately omitted.

The particle analysis device of the present technology is a device including a chamber that has at least one well or through hole and that includes a microchip that divides into a first space and a second space. Furthermore, the particle analysis device of the present technology includes a pressurizing step of applying a positive pressure to the fluid in the chamber, and a first flow path connected to the first space and/or a first flow path connected to the second space. The valve opening/closing step of operating a valve for opening/closing the two flow paths is performed to discharge bubbles in the chamber. The particle analysis device of the present technology includes a pressure control unit that controls the pressurizing process and the valve opening/closing process.
Furthermore, the particle analysis device according to an embodiment of the present technology includes an observation unit that images a bubble in the chamber with a microscope, and an analysis unit that analyzes the bubble based on information acquired from the observation unit. Prepare And/or, the particle analysis device according to the present technology, an observation unit that images a particle captured in the well or the through hole with a microscope, and based on the information acquired from the observation unit, the captured particle And an analysis unit for performing analysis regarding.

The time for performing the bubble discharging method of the present technology may be before supplying the particles into the chamber or after supplying the particles.

In the particle analysis device of the present technology, bubbles existing in the fluid in the chamber can be efficiently discharged by the pressure control unit, the analysis unit, and the like. This makes it possible to improve the accuracy and efficiency of particle analysis and the like. For example, when counting particles such as cells from the shape by image analysis such as imaging, false positive results will occur if air bubbles of the same size as particles such as cells are mixed. The fact that air bubbles can be discharged can reduce, prevent or avoid such a situation. For example, when capturing particles such as cells, compared to the case where air bubbles are mixed in a particle imaging image of cells after capturing particles such as cells, the present technology allows the mixed air bubbles to be more discharged. Good contrast is obtained.
Note that the effects produced by the present technology are not necessarily limited to the effects described here, and may be any effects described in the present specification.

<2-5. Example 1 of device according to the present technology>
An example of the device according to the present technology (see, for example, FIG. 2, FIG. 6, and FIG. 7) is given, but the present technology is not limited thereto.
The device of the present technology includes a chamber 100 that has at least one well 2 and that includes a microchip that divides into a first space 3 and a second space 4. The chamber 100 preferably includes the pressure control unit 20. The well 2 may be provided with a hole 8 in the well bottom portion 7, and the well 2 may be a through hole, and the well 2 or the through hole may be connected to the first space 3 and the second space 4. Preferably. It is preferable that the well 2 or the through hole has a size (or space) that allows only one particle to enter. Further, it is preferable that the chamber 100 has a region 9 including at least one well 2 or a through hole (hereinafter, also referred to as “well/through hole region 9”).

The space in the chamber 100 of the present technology is divided into the first space 3 and the second space 4 by the well/through hole region 9.

The first space 3 is connected to the first flow paths 5 and 5. Each of the first flow paths 5 and 5 is preferably connected to a first supply valve 51 and a first discharge valve 52, respectively. By opening and closing the first valves 51, 52 connected to the first flow path 5, the discharge and supply of the fluid to the first space 3 can be controlled. For convenience, the first supply valve 51 is used as a supply valve and the first discharge valve 52 is used as a discharge valve, but the first valves 51 and 52 can be appropriately changed to supply or discharge.

The second space 4 is connected to the second flow paths 6 and 6. Each of the second flow paths 6 and 6 is preferably connected to the second supply valve 61 and the second discharge valve 62, respectively. By opening/closing the second valves 61, 62 connected to the second flow path 6, the discharge and supply of the fluid to the second space 4 can be controlled. For convenience, the second supply valve 61 is used as a supply valve and the second discharge valve 62 is used as a discharge valve. However, the second valves 61 and 62 can be appropriately changed to supply or discharge.

The first flow path 5 may be connected to a flow path for supplying a fluid and/or a flow path for discharging a fluid via the first supply valve 51 or the first discharge valve 52.
Further, the second flow path 6 may be connected to the flow path for supplying the fluid and/or the flow path for discharging the fluid via the second supply valve 61 or the second discharge valve 62.
Further, the flow channel for supplying the fluid and/or the flow channel for discharging the fluid may be connected to the first space 3 or the second space 4 via the first flow channel 5 or the second flow channel 6, It may be connected directly to the first space 3 or the second space 4. The fluid may contain particles.

The pressure control unit 20 has at least one well 2 or a through hole and is configured to discharge bubbles in the chamber 100 including a microchip that divides the first space 3 and the second space 4. Has been done. The pressure control unit 20 includes a pressurizing step of applying a positive pressure to the fluid in the chamber, and a first flow path 5 connected to the first space 3 and/or a first flow path connected to the second space 4. A valve opening/closing step of operating valves 51, 52, 61, 62 for opening/closing the two flow paths 6 is performed, and control is performed to discharge bubbles in the chamber. Further, the pressure control unit 20 causes the above-mentioned <1. The bubble discharge method according to the present technology> can be performed, and the device of the present technology is configured to perform the bubble discharge method of the present technology.

Further, it is preferable that the device of the present technology is capable of capturing particles in the chamber. In this case, the well/through hole region 9 functions as a particle capturing region for capturing particles, and the particles can be captured in the well 2 or the through holes. Further, as the device of the present technology, a particle capturing device or a particle analyzing device is suitable.
The device according to the present technology preferably includes a suction unit (not shown) that suctions the fluid or the fluid containing particles existing in the first space 3 or the second space 4. The suction unit can be directly or indirectly connected to the first space 3 and/or the second space 4. When the suction unit is indirectly connected to the first space 3 or the second space 4, for example, the suction unit may be provided via the first flow path 5 and/or the second flow path 6.
By the suction part, the particles can be sucked to the side opposite to the settling side of the particles, and the speed of the settling of the particles can be adjusted.

The chamber 100 of the present technology may be configured so that the particles captured in the well 2 can be observed using an imaging device (not shown) such as the microscope 80. For example, it is preferable that the chamber is made of a transparent material so that one surface of the chamber can be observed. Thus, by configuring at least a part of the chamber with a transparent material, the captured particles can be observed by an observation unit such as a microscope 80 (for example, an upright microscope or an inverted microscope). The observation unit such as the microscope 80 allows observation of the well 2 or the through holes, particles S, bubbles, etc. existing in the channel from the upper surface or the lower surface of the well/through hole region 9 of the chamber.

The observation unit may further include an imaging device (not shown). Examples of the image pickup device include an image pickup device provided with an image sensor, particularly a digital camera. The image sensor can be, for example, a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor). The image data obtained by the image capturing may be stored in the image capturing device or the analyzing unit, or may be stored in an external data storage device connected to the image capturing device by wire or wirelessly.

In this technology, bubbles existing in the fluid in the chamber can be efficiently discharged. This makes it possible to improve the accuracy and efficiency of particle analysis and particle trapping in an apparatus or system for particle analysis, particle trapping, particle analysis, etc. using a flow channel system. Note that the effects produced by the present technology are not necessarily limited to the effects described here, and may be any effects described in the present specification.

The particles captured in the well can be subjected to various observations and/or measurements. For example, a predetermined fluorescent label may be attached to the particles before the particles are supplied into the chamber, and the particles that emit the strongest fluorescence after the particles are captured can be selected from the captured particles. Furthermore, only the selected particles can be taken out from the chamber for capturing particles by a single particle acquisition device such as a micromanipulator. Then, another process is performed using the selected particles. When the particle is a cell, the other treatment may be, for example, genetic analysis, culturing, and substance production.
In addition, it is possible to select particles having desired characteristics such as selection of cells that carry out desired antibody secretion, selection of cells or microorganisms that carry out desired gene expression, and selection of cells having desired differentiation ability. ..

<2-6. Example 2 of device according to the present technology>
Example 2 of another aspect of the device according to the present technology will be described with reference to FIG. 9. FIG. 9 is a block diagram of an example of the device of the present technology.
As shown in FIG. 9, a device 1200 according to an embodiment of the present technology includes a particle capturing chamber 1201, a suction unit 1202, a fluid supply unit 1203, a fluid recovery unit 1204, an observation unit 1205, a control unit 1206, and an analysis unit 1207. The unit 1206 includes a pressure control unit (not shown). The device of the present technology is not limited to this.

The particle trapping chamber 1201 includes a particle trapping region having at least one well or a through hole, and a particle trapping channel portion used for trapping particles in the well or in the through hole. .. The particle capturing chamber 1201 is further provided with a fluid supply flow path section and a fluid discharge flow path section.

In the present technology, the particle trapping area may be replaceable. The particle-trapping chip in the particle-trapping chamber 1201 may be detachably provided from the chamber. Then, for example, the particle-capturing chip may be replaced by the user for each analysis.
Alternatively, the particle capture chamber 1201 itself may be replaceable. That is, the apparatus 1200 according to the present technology may be detachably provided with the particle capturing chamber 1201. For example, according to the present technology, a cartridge-shaped particle capturing chamber unit in which a particle capturing chip and a chip holder holding the chip are integrated may be replaceably provided in the device 1200 according to the present technology. In this case, since the user can replace the particle capturing area by replacing the cartridge, it is easier to handle than replacing only the particle capturing area, which is a small thin film. Further, in this case, since the inside of the chamber is not exposed, it is possible to prevent dust from adhering to the well.

The suction unit 1202 can suction particles in the chamber through the first flow path and the second flow path of the particle capturing chamber 1201. For example, suction is performed in the particle capturing step described above. The suction unit 1202 can be connected to the particle capturing chamber 1201 (for example, the first space or the second space, the first channel or the second channel) so that the suction can be performed. In this connection, for example, a tube for performing suction may be connected. A valve may be provided on the tube. The suction unit includes, for example, a pump.

The fluid supply unit 1203 supplies a fluid containing particles to the particle capturing chamber 1201. For example, in the above-mentioned particle capturing step, it is used for supplying a fluid containing particles into the particle capturing chamber by supplying or suctioning. The fluid supply unit includes, for example, a container capable of containing a fluid containing particles and a pipe connected to the container. The tube can communicate with the particle capturing chamber 1201 (for example, the first space or the second space, the first flow path or the second flow path). A valve may be provided on the tube.

The fluid recovery unit 1204 recovers the fluid from the particle capturing chamber 1201. For example, the fluid recovery unit 1204 removes particles in the above-described removal process. The fluid recovery unit 1204 may be connected to the particle capturing chamber 1201 so that the fluid can be recovered from the particle capturing chamber 1201. For example, the particle capturing chamber 1201 (for example, the first space or the second space, the first flow path or the second flow path, etc.) and the pipe for performing the fluid recovery of the fluid recovery unit 1204 can be communicated with each other. A valve may be provided on the tube. The fluid recovery unit 1204 may include, for example, a pump. The fluid in the chamber is collected by the suction by the pump. For example, the fluid recovery unit 1204 can be connected to the particle capturing chamber 1201 via a liquid recovery container so that the liquid sucked by the fluid recovery unit does not enter the pump.

The fluid recovery unit 1204 may be provided in one, two, or three or more in the particle capturing chamber 1201. For example, if two fluid recovery parts are provided in the particle capturing chamber 1201, one fluid recovery part is used to recover particles not captured in the well or in the through-hole, and another fluid recovery part. A collector can be used to collect particles trapped in the wells or through holes.

The observation unit 1205 is used for observing particles trapped in the well or in the through hole and/or grasping characteristics of particles trapped in the well or in the through hole. Further, the observation unit 1205 may be used to grasp the mixed bubbles in the chamber.
Further, the observation of particles may include, for example, observation of the shape, structure, and/or color of the particles themselves. The grasping of the characteristics relating to the particles may include grasping the wavelength and/or the intensity of light emitted from the particles, such as fluorescence.
Further, the observation of the mixed bubbles may include, for example, the observation of the size, number, and movement of the bubbles themselves. The observation of the mixed bubbles may be performed by focusing on the bubbles or by imaging the bubbles. Further, the observation of the bubbles may be grasped by capturing the image after capturing and analyzing the size, number and movement of the bubbles in the image.

The observation unit 1205 may be, for example, a device that enables the observation and/or the grasp, and may be, for example, a microscope and/or a photodetector, but microscope observation is preferable when observing bubbles. The observation of particles and the observation of bubbles may be performed by providing different devices, or may be performed by the same device.
In the present technology, it is configured to be arranged at a position where bubbles and particles can be observed in the chamber. For example, the observation unit 1205 may be provided above and/or below the particle capturing chamber 1201. For example, it is preferable to use an inverted microscope as the microscope. Also preferably the microscope may be a light microscope. That is, in the present technology, the observation unit 1205 preferably includes an inverted optical microscope.

The generally adopted bright-field observation or dark-field observation for observing the appearance characteristics of cells may be adopted in the present technology. Further, when observing cells in a state in which the fine internal structure of transparent cells is emphasized, phase contrast observation or differential interference observation suitable for such a case may be adopted in the present technology. By adopting these observation methods, it is possible to observe cells in a living state without staining them. For observation of transparent cells, it is particularly preferable to adopt phase contrast observation. When the phase difference observation is adopted, it is preferable that the observation unit 1205 includes a halogen lamp light source, an objective lens, a phase plate, a condenser lens, and a ring diaphragm.

Also, if cells are labeled with a fluorescent protein, it is possible to observe the cells by fluorescence observation while highlighting a specific part of interest in the cells. Such fluorescence observation is used in various applications such as identification of an antigen by an antigen-antibody reaction or visualization of intracellular structures such as mitochondria. When performing fluorescence observation, the observation unit 1205 cuts off a light source for excitation (generally a mercury lamp), a filter for selecting the wavelength of excitation light, a dichroic mirror for extracting fluorescence of a wavelength emitted by a substance, and a wavelength other than the fluorescence wavelength. It is preferred to include an absorption filter. By selecting a combination of excitation wavelength and fluorescence wavelength with a filter, it is possible to perform various analyzes from one observation image.

The observation unit 1205 may further include an imaging device. Examples of the image pickup device include an image pickup device provided with an image sensor, particularly a digital camera. The image sensor can be, for example, a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor). The image data obtained by imaging may be stored in the imaging device or the analysis unit 1207, or may be stored in an external data storage device connected to the imaging device by wire or wirelessly.

The control unit 1206 may control the suction unit 1202, the fluid supply unit 1203, and/or the fluid recovery unit 1204. For example, the pump and/or valve of the suction unit 1202, the fluid supply unit 1203, and/or the fluid recovery unit 1204 may be controlled. Thereby, various steps in the particle capturing method or particle analyzing method of the present technology can be performed.
In addition, the control unit 1206 may control the pressure control unit, or may include the pressure control unit inside or outside. Thereby, various steps in the bubble discharging step of the present technology, for example, a pressurizing step, a valve opening/closing step, a bubble analyzing step, etc. can be performed.

The analysis unit 1207 analyzes the data obtained by the observation unit 1205, such as image data or light-related data. For example, the analysis unit 1207 can also perform the analysis in the analysis step in the bubble discharging method and the analysis step in the particle capturing method or the particle analyzing method described above.
The analysis unit 1207 can select, for example, particles having a predetermined shape or color or mixed bubbles based on the obtained image data. Alternatively, in the case of particles, particles that emit a predetermined fluorescence can be selected based on the obtained data regarding light. The position information regarding the selected particles and the mixed bubbles can be transmitted from the analysis unit 1207 to a device of the present technology, such as a single particle capturing device (for example, a micromanipulator), which is connected by wire or wirelessly. Based on the position information of the mixed bubbles, the movement, size and number of the mixed bubbles can be grasped and used for discharging the bubbles. In addition, the selected particle can be acquired alone by the single particle capturing device based on the position information of the particle.

The device 1200 may be provided with other components in addition to the components described above, such as the above-mentioned single particle capturing device. In addition, the device 1200 stores, as necessary, a storage unit for storing various data, an input unit for inputting an instruction regarding trapped bubbles and particles from a user, and an observation or analysis result of the trapped bubbles, An output unit for outputting various results such as a capture result and an analysis result may be provided.

Note that the present technology may also be configured as below.
[1]
In a chamber including a microchip having at least one well or through hole and dividing into a first space and a second space,
A pressurizing step of applying a positive pressure to the fluid in the chamber,
A valve opening/closing step of operating a valve for opening/closing a first flow path connected to the first space and/or a second flow path connected to the second space,
Bubble discharge method.
[2]
The bubble discharging method according to [1], wherein the microchip is a particle trapping chip having a particle trapping region including at least one well or through hole.
[3]
The first flow path is a flow path connected to a first supply valve to which a fluid is supplied and a first discharge valve to which a fluid is discharged, and/or
The bubble discharging method according to [1] or [2], wherein the second flow path is a flow path connected to a second supply valve for supplying a fluid and a second discharge valve for discharging the fluid.
[4]
The valve opening and closing step includes a step of opening and closing the supply valve and/or the discharge valve after applying a positive pressure to the fluid in the chamber to discharge bubbles from the chamber. Bubble discharge method.
[5]
The bubble discharging method according to any one of [1] to [4], wherein the pressurizing step and the valve opening/closing step are repeated.
[6]
The bubble discharging method according to any one of [1] to [5], further including a bubble analysis step of analyzing bubbles in the chamber based on information obtained by imaging the bubbles in the chamber.
[7]
In the bubble analysis step, it is determined whether or not the bubbles in the chamber meet a predetermined condition, and when it is determined that the bubbles meet the predetermined condition, the pressurizing step and the valve opening/closing step are performed. 6] The bubble discharging method described above.
[8]
In the pressurizing step, the supply valve is opened, and a valve other than the supply valve is closed to pressurize the fluid in the chamber,
The bubble discharging method according to any one of [2] to [7], wherein in the valve opening/closing step, after closing the supply valve, any one or more of the closed valves are opened intermittently.
[9]
In the pressurizing step, the supply valve is opened, and a valve other than the supply valve is closed to pressurize the fluid in the chamber,
The bubble discharging method according to any one of [2] to [7], wherein any one or more of the closed valves are opened in the valve opening/closing step.
[10]
The bubble discharging method according to [2], wherein the particles are single cells.

[11]
In a chamber having a microchip having at least one well or through hole and partitioning into a first space and a second space,
A pressurizing step of applying a positive pressure to the fluid in the chamber,
And a valve opening/closing step of operating a valve for opening/closing a first flow path connected to the first space and/or a second flow path connected to the second space, and discharging bubbles in the chamber. It is equipped with a pressure control unit, which controls
A particle capturing device for capturing particles in the well or the through hole.
[12]
The particle capturing device according to [11], wherein the microchip is configured such that an upper portion of the first space becomes higher in a discharging direction.
[13]
The particle capturing device according to [12], wherein the microchip is configured such that a cross-sectional area in the width direction increases in the discharging direction.
[14]
The particle capture device according to any one of [11] to [13], wherein the pressure control unit executes any one of the bubble discharging methods according to [3] to [9].

[15]
In a chamber having a microchip having at least one well or through hole and partitioning into a first space and a second space,
A pressurizing step of applying a positive pressure to the fluid in the chamber,
And a valve opening/closing step of operating a valve for opening/closing a first flow path connected to the first space and/or a second flow path connected to the second space, and discharging bubbles in the chamber. Pressure control unit, which controls
An observation unit that images a bubble in the chamber and/or particles captured in the well or the through hole with a microscope, and
An analysis unit that performs analysis on the bubbles and/or the trapped particles based on the information acquired from the observation unit,
A particle analysis device comprising:
[16]
The pressure control unit and the observation unit are controlled by a central control unit, and the central control unit executes the bubble discharging method according to any one of the above [3] to [9]. apparatus.

2 well 3 1st space 4 2nd space 5 1st flow path 6 2nd flow path 7 well bottom 8 hole 9 well/through hole area 10 microchip 20 pressure controller, pressure control unit 51 first supply valve 52 first discharge Valve 61 Second supply valve 62 Second discharge valve P Pump 70 Container 71 Particle flow path 72 Particle supply valve 80 Microscope, inverted microscope 100 Chamber 1200 Device 1201 Particle capture chamber 1202 Suction part 1203 Fluid supply part 1204 Fluid recovery part 1205 Observation unit 1206 Control unit

Claims (14)

In a chamber including a microchip having at least one well or through hole and dividing into a first space and a second space,
A pressurizing step of applying a positive pressure to the fluid in the chamber,
A valve opening/closing step of operating a valve for opening/closing a first flow path connected to the first space and/or a second flow path connected to the second space,
Bubble discharge method. The method for discharging bubbles according to claim 1, wherein the microchip is a particle capturing chip having a particle capturing region including at least one well or a through hole. The first flow path is a flow path connected to a first supply valve to which a fluid is supplied and a first discharge valve to which a fluid is discharged, and/or
The bubble discharging method according to claim 1, wherein the second flow path is a flow path connected to a second supply valve for supplying a fluid and a second discharge valve for discharging the fluid. 4. The valve opening/closing step according to claim 3, further comprising the step of opening/closing the supply valve and/or the discharge valve after applying a positive pressure to the fluid in the chamber to discharge bubbles from the chamber. Bubble discharge method. The bubble discharging method according to claim 1, wherein the pressurizing step and the valve opening/closing step are repeated. The bubble discharging method according to claim 1, further comprising a bubble analyzing step of analyzing bubbles in the chamber based on information obtained by imaging the bubbles in the chamber. In the bubble analysis step, it is determined whether or not the bubbles in the chamber meet a predetermined condition, and when it is determined that the bubbles meet the predetermined condition, the pressurizing step and the valve opening/closing step are performed. 6. The bubble discharging method described in 6. In the pressurizing step, the supply valve is opened, and a valve other than the supply valve is closed to pressurize the fluid in the chamber,
The bubble discharging method according to claim 3, wherein in the valve opening/closing step, after closing the supply valve, at least one of the closed valves is opened intermittently. In the pressurizing step, the supply valve is opened, and a valve other than the supply valve is closed to pressurize the fluid in the chamber,
The bubble discharging method according to claim 3, wherein in the valve opening/closing step, any one or more of the closed valves are opened. The method for discharging bubbles according to claim 2, wherein the particles are single cells. In a chamber containing a microchip having at least one well or through hole and partitioning into a first space and a second space,
A pressurizing step of applying a positive pressure to the fluid in the chamber,
And a valve opening/closing step of operating a valve for opening/closing a first flow path connected to the first space and/or a second flow path connected to the second space, and discharging bubbles in the chamber. Is equipped with a pressure control unit,
A particle capturing device for capturing particles in the well or the through hole. The particle capturing device according to claim 11, wherein the microchip is configured such that the upper part of the first space becomes higher in the discharging direction. The particle capturing device according to claim 12, wherein the microchip is configured such that the cross-sectional area in the width direction increases in the discharging direction. In a chamber containing a microchip having at least one well or through hole and partitioning into a first space and a second space,
A pressurizing step of applying a positive pressure to the fluid in the chamber,
And a valve opening/closing step of operating a valve for opening/closing a first flow path connected to the first space and/or a second flow path connected to the second space, and discharging bubbles in the chamber. Pressure control unit, which controls
An observation unit that images a bubble in the chamber and/or particles captured in the well or the through hole with a microscope, and
A particle analysis device, comprising: an analysis unit that performs analysis on the bubbles and/or the trapped particles based on information acquired from the observation unit.

Images